The goal of this Phase II Proposal is the development of a simple, cost- effective, and high-throughput method for detecting RNA directly and quantitatively. The advent of genome-wide sequencing projects and expression array analysis has led to an unprecedented demand for an accurate method to measure specific gene expression that can accommodate large numbers of samples. This Phase II Proposal describes the further development of an enzymatic assay for direct and quantitative analysis of RNA in a microtiter plate-based format. The assay relies on structure-specific cleavage of oligonucleotides hybridized to the target RNA strand, and can be adapted to any sequence. During the Phase I Project, we successfully increased the sensitivity of the detection to about 10/5 copies, through the use of solid phase capture and ELISA detection of the cleavage products, allowing direct detection of common mRNAs from the cell. In this Phase II Proposal, we propose to use novel chimeric enzymes and reaction modifications to drive the sensitivity down to levels suitable for detection of rare mRNAs, at least a 10-fold, and ideally a 100- fold improvement. The assay will be able to quantitate mRNAs from tissue culture cells without prior RNA purification in about 4 hours. The assay will also be configured to use a fluorescence resonance energy transfer readout, eliminating post-reaction sample processing, reducing variation, and making the assay more suitable for high-throughput screening. PROPOSED COMMERCIAL APPLICATION: This project could result in a low-cost, high-throughput, microtiter plate- based method for the direct detection of RNA for monitoring gene expression of a wide variety of genes. This versatile technology could capture a significant portion of the $100 million research market for monitoring cytokine expression, and see broad application in expression screening for drug development. In addition, the enhanced detection may improve capability for mRNA quantitation.